For many biological, biochemical, diagnostic or therapeutic purposes, it is necessary to accurately determine the amount or concentration of a nucleic acid in a sample. For this purpose usually external and/or internal standards are used. It is assumed that the standard has the “true concentration” which is precisely known. This may not be true and can usually not be easily verified if a relatively inaccurate method such as UV absorption or real-time PCR is used. The concentration of standards often relies on a chain of calibration standards traced back to the e.g. a WHO standard, hence quantitation errors often pile up. Quantitation standards are often imprecise, because they refer to one or several secondary standards produced and stored by a company, which again refer to another standard, e.g. the WHO standard. Even if the reference chain is properly done and accurate, the final standard may have degraded or subject to a production error, which is hard to verify unless very stringent quality control is used.
Even with methods allowing for accurate quantification of a nucleic acid in a sample tested, it is usually only possible to exactly determine the concentration of the purified nucleic acid rather than the unprocessed nucleic acid in the reaction mixture which is used as input for quantification reaction. What is however of real interest, especially in medical diagnostics applications such as initial diagnosis or disease or therapy monitoring (e.g. medical decision points in minimal residual disease monitoring) is the target concentration in the primary or unprocessed sample. The amount of nucleic acid which makes its way from the primary sample (e.g. plasma from human blood) to the quantification reaction is poorly known, because of pipetting errors and the unknown efficiency of the sample preparation process.
Therefore, it is a common practice to add a quantitation standard to the primary sample to track all losses along the preparation process. However, the state of the art is to rely on the accuracy of the quantitation standard, i.e. neglecting the errors described above. Further, it is difficult and expensive to provide a suitable external standard (e.g. a commercially available standard) in a concentration which is exactly known. Moreover, it needs to be routinely verified by a chain of secondary and tertiary standards, which in turn need to be checked on a regular basis.
Accordingly, there is a need for methods of quantifying a nucleic acid of interest, which avoids the above disadvantages, particularly which does not require a standard of known concentration.